The skin is a continually renewing tissue consisting of a large population of transit amplifying (TA) cells with a limited proliferative potential, and a smaller population of keratinocyte stem cells (KSCs) that have a high proliferative potential and are clonogenic. KSCs renew the stem cell population and give rise to TA cells, which are displaced to the suprabasal layers and are lost by terminal differentiation. In the skin, the stem cell population resides in the hair follicle bulge, which is located in the permanent portion of the hair follicle and is protected from both physical damage and the changes the hair follicle undergoes as it cycles from resting (telogen) to active growth (anagen). In the well-known two- stage murine epidermal carcinogenesis model, it is a widely held belief that KSCs are the primary carcinogen target cells (i.e., latent neoplastic cells). A key factor supporting this belief is the fact that DMBA-initiated mice will develop skin tumors upon exposure to a tumor promoter such as TPA whether it is applied a week or a year after initiation. Given that the keratinocyte population renews itself every 6 days in the mouse, the fact that initiated cells persist suggests that these must be slowly cycling cells located in a protected microenvironment. Our current focus is on identification and characterization of the cells that give rise to cutaneous neoplasms in the mouse, as well as investigation into signaling pathways that contribute to neoplastic development. As part of this objective, we have investigated the hematopoietic stem and progenitor cell marker, CD34, in the skin and using this marker in combination with alpha-6 integrin and fluorescence activated cell sorting (FACS), have shown that CD34 specifically marks hair follicle bulge keratinocytes, and facilitates isolation of live follicular bulge keratinocytes that represent a subset of alpha-6 integrin bright cells that are quiescent (i.e., predominantly in G1/G0). This work represents the first use of a bulge-specific cell surface marker allowing for direct positive enrichment of live keratinocyte stem and progenitor cells. Recently we made a comparison was made between CD34+ keratinocytes harvested from either TPA-treated or untreated Tg.AC mice to investigate differential gene expression patterns following tumor promotion. Using nylon cDNA arrays from Clontech probed with PCR-based SMART-amplified cDNA prepared from CD34+ cells isolated from TPA-treated or untreated skin, eleven genes were identified whose expression changed significantly in response to treatment with TPA. Of particular interest was Deleted in Split Hand/Split Foot 1 (Dss1), which is associated with a heterogeneous limb developmental disorder. Overexpression of Dss1 and NDPK-B was detected by RT-PCR and Northern analysis in TPA treated skin (non-tumor bearing; compared to low levels in untreated skin), as well as in cutaneous tumors, including papillomas, squamous cell carcinomas, and spindle cell tumors. Functional studies revealed an increase in foci-forming activity and proliferation of preneoplastic epidermal cells constitutively expressing Dss1. Interestingly, Dss1 induced transformation of stably transfected JB6 epidermal cells was abrogated by addition of a protein kinase C (PKC) specific inhibitor, implicating a possible PKC regulatory role in Dss1 expression. Taken, together, these results suggest that Dss1 is a TPA-inducible gene that may play an important role in the early stages of skin carcinogenesis. In addition, recent studies have implicated a potential involvement of NDBK-B in early-stage neoplastic development in chemically-induced skin carcinogenesis. In addition, we have furthered these experiments utilizing high density microarray technology, comparing gene expression profiles developed from CD34+ and CD34- cells isolated from TPA treated and acetone control treated Tg.AC and wild type FVB/N mice. From this, comparing back to datasets generated from untreated mice, we have developed a picture of early response genes differentially affected in epidermal stem cells. To understand key signaling pathways in tumor development, we are exploring the relationship between Ha-ras and p19ARF in benign and malignant tumor development using a bigenic mouse strain made by crossing p19ARF-null and Tg.AC mice. Initial findings in skin tumorigenesis experiments supported a role for p19ARF in tumor development, but in the course of these studies, an unexpected tumor type developed in the bigenic mice, Gastrointestinal Stromal Tumors (GIST), which are highly aggressive tumors in humans, and which developed spontaneously at a high incidence in bigenic mice, making this a potential mouse model for this tumor type. Finally, to assess further the role of the hair follicle epidermal stem cell population in cutaneous tumor development, we have utilized the technique of epidermal abrasion, in which the interfollicular epidermis is physically removed. The resulting epidermal regeneration is derived from keratinocytes migrating out from the underlying hair follicles. Expression profiles using high-density microarray analysis were prepared from samples collected at days 3, 5, 9, 18, and 30 post-abrasion from abraded FVB/N (wild type) and Tg.AC mice (which develop ras-transgene dependent papillomas following a single abrasion). We have developed bioinoformatics strategies designed to probe this complex dataset in terms of global analysis, allowing for more informed focus on smaller subsets of genes that may contribute to tumor development and epidermal regeneration.